Characterization of two genes encoding distinct transferrin-binding proteins in different Actinobacillus pleuropneumoniae isolates.
ABSTRACT: The gene encoding the Actinobacillus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70% of the protein was expressed as an aggregate protein in Escherichia coli. The nucleotide sequences of the tfbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that of A. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The tfbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tfbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.
Project description:The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A.A. Potter, and P.J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate.
Project description:Actinobacillus pleuropneumoniae can use porcine transferrin as the sole source of iron. Two proteins with molecular masses of approximately 60 kDa (TfbA) and 110 kDa have been shown to specifically bind porcine transferrin; from the TfbA protein, three isoforms from A. pleuropneumoniae serotypes 1, 5, and 7 have been identified and characterized by nucleotide sequence analysis. Here we defined the transferrin-binding region(s) of the TfbA protein of A. pleuropneumoniae serotype 7 by TnphoA mutagenesis, random mutagenesis, and peptide spot synthesis. The amino-terminal half of the TfbA molecule, which has only 36% amino acid sequence identity among the three isoforms, was shown to be responsible for transferrin binding by TnphoA mutagenesis. This result was confirmed by analysis of six random mutants with decreased transferrin binding affinity. The subsequent analysis of overlapping 16-mer peptides comprising the amino-terminal half of the TfbA molecule revealed three domains of 13 or 14 amino acids in length with transferrin-binding activity. They overlapped, or were very close to, point mutations decreasing transferrin-binding ability. The first and third domains were unique to the TfbA protein of A. pleuropneumoniae serotype 7. In contrast, the sequence of the second domain was present in almost identical forms (12 of 14 residues) in the TfbA proteins of A. pleuropneumoniae serotypes 1 and 5; in addition, a sequence consisting of functionally homologous amino acids was present in the otherwise completely distinct small transferrin-binding proteins of Neisseria gonorrhoeae (TbpB), N. meningitidis (Tbp2), and Haemophilus influenzae (Tbp2).
Project description:Previous studies have shown that a vaccine prepared from outer membranes of Actinobacillus pleuropneumoniae serotype 5 can elicit protective immunity in swine against challenge with either serotype 5 or serotype 1. These results suggest the presence of common subcapsular surface antigens, such as outer membrane proteins, that contribute to cross-protective immunity. We have identified a 48-kDa outer membrane protein that is common to all 12 capsular serotypes of A. pleuropneumoniae but is not present in the outer membranes of related species of gram-negative swine pathogens. This protein is immunogenic in swine infected with either A. pleuropneumoniae serotype 5 or 1A, as well as in swine vaccinated with A. pleuropneumoniae serotype 5 outer membranes. This 48-kDa protein is readily detected in outer membranes produced by sucrose density gradient centrifugation, but it is sarcosyl soluble and therefore not found in outer membranes prepared by detergent treatment. The gene encoding the 48-kDa outer membrane protein has been cloned from A. pleuropneumoniae serotype 5 and and has been designated aopA, for Actinobacillus outer membrane protein A. The gene is 1,347 bp in length and encodes a protein, designated AopA, of 449 amino acids with a predicted molecular weight of 48,603. Southern blot analysis under high-stringency conditions showed that strains of all 12 serotypes of A. pleuropneumoniae contain DNA homologous to this gene, as do strains of two closely related species, Actinobacillus suis and Pasteurella multocida. Whether antibodies against the AopA antigen contribute to cross-protective immunity against A. pleuropneumoniae infection remains to be determined.
Project description:Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15.
Project description:RTX cytolysins are a family of calcium-dependent, pore-forming, secreted toxins found in a variety of gram-negative bacteria. The prototypical member of this family is the alpha-hemolysin of Escherichia coli. The RTX genetic determinants from seven members of the family Pasteurellaceae, Pasteurella haemolytica, Actinobacillus actinomycetemcomitans, and A. pleuropneumoniae serotypes 1,5,7, and 9 were previously cloned and sequenced. Using the leukotoxin determinant from P. haemolytica serotype A1 as a probe, we detected the presence of RTX-type determinants in Actinobacillus suis, A. equuli, and A. lignieresii of the family Pasteurellaceae. All three species elaborate proteins of approximately 104 to 110 kDa that are recognized by polyclonal antisera against the 104-kDa hemolysin of A. pleuropneumoniae serotype 1. An RTX determinant of A. suis isolate 3714 was cloned and sequenced and was found to be almost identical to the RTX determinant of A. pleuropneumoniae serotypes 5 and 9. In addition, the determinant is not composed of four contiguous genes, as had been reported for most other RTX determinants; instead, the genes encoding the two proteins responsible for secretion of the toxin are at a locus distinct from that containing the toxin structural and activation genes.
Project description:The genetic organization of the gene involved in the capsular polysaccharide (CPS) biosynthesis of Actinobacillus pleuropneumoniae serotype 14 has been determined. The DNA region for the CPS biosynthesis of serotype 14 (cps14) comprised 9 open reading frames, designated as cps14AB1B2B3CDEFG genes, encoding Cps14A to Cps14G protein, respectively. Cps14A was similar to CpsA of A. pleuropneumoniae serotypes 1, 4 and 12; the Cps14B1 and Cps14B2 were similar to CpsB of A. pleuropneumoniae serotypes 1, 4 and 12, suggesting that CPS structure of A. pleuropneumoniae serotype 14 would belong to Group I including A. pleuropneumoniae serotypes 1, 4, 12 and 15. Surprisingly, the overall nucleotide sequence, deduced amino acid sequence, and the genetic organization of the cps14 were nearly identical to those of Actinobacillus suis. This study will provide the molecular basic knowledge for development of diagnostics and vaccine of A. pleuropneumoniae serotype 14.
Project description:BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia, a highly contagious respiratory infection in pigs, and all the 15 serotypes are able to cause disease. Current vaccines including subunit vaccines could not provide satisfactory protection against A. pleuropneumoniae. In this study, the immunoproteomic approach was applied to the analysis of extracellular and outer membrane proteins of A. pleuropneumoniae JL03 serotype 3 for the identification of novel immunogenic proteins for A. pleuropneumoniae. RESULTS: A total of 30 immunogenic proteins were identified from outer membrane and extracellular proteins of JL03 serotype 3, of which 6 were known antigens and 24 were novel immunogenic proteins for A. pleuropneumoniae. CONCLUSION: These data provide information about novel immunogenic proteins for A. pleuropneumoniae serotype 3, and are expected to aid in development of novel vaccines against A. pleuropneumoniae.
Project description:An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping. One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis. It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A. pleuropneumoniae. This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA. Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A. pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains. Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 1 isolate.
Project description:BACKGROUND:Protection of pigs by vaccination against Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is hampered by the presence of 15 different serotypes. A DIVA subunit vaccine comprised of detergent-released proteins from A. pleuropneumoniae serotypes 1, 2 and 5 has been developed and shown to protect pigs from clinical symptoms upon homologous and heterologous challenge. This vaccine has not been characterized in-depth so far. Thus we performed i) mass spectrometry in order to identify the exact protein content of the vaccine and ii) cross-serotype 2-D immunoblotting in order to discover cross-reactive antigens. By these approaches we expected to gain results enabling us to argue about the reasons for the efficacy of the analyzed vaccine. RESULTS:We identified 75 different proteins in the vaccine. Using the PSORTb algorithm these proteins were classified according to their cellular localization. Highly enriched proteins are outer membrane-associated lipoproteins like OmlA and TbpB, integral outer membrane proteins like FrpB, TbpA, OmpA1, OmpA2, HgbA and OmpP2, and secreted Apx toxins. The subunit vaccine also contained large amounts of the ApxIVA toxin so far thought to be expressed only during infection. Applying two-dimensional difference gel electrophoresis (2-D DIGE) we showed different isoforms and variations in expression levels of several proteins among the strains used for vaccine production. For detection of cross-reactive antigens we used detergent released proteins of serotype 7. Sera of pigs vaccinated with the detergent-released proteins of serotypes 1, 2, and 5 detected seven different proteins of serotype 7, and convalescent sera of pigs surviving experimental infection with serotype 7 reacted with 13 different proteins of the detergent-released proteins of A. pleuropneumoniae serotypes 1, 2, and 5. CONCLUSIONS:A detergent extraction-based subunit vaccine of A. pleuropneumoniae was characterized by mass spectrometry. It contained a large variety of immunogenic and virulence associated proteins, among them the ApxIVA toxin. The identification of differences in expression as well as isoform variation between the serotypes implied the importance of combining proteins of different serotypes for vaccine generation. This finding was supported by immunoblotting showing the induction of cross-reactive antibodies against several surface associated proteins in immunized animals.
Project description:Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.